Meta-analysis of clinical risk factors of patients with systemic lupus erythematosus complicated with invasive fungal infection

Background: To systematically evaluate the clinical risk factors of patients with systemic lupus erythematosus (SLE) complicated with invasive fungal infection (IFI) among patients. Methods: A meta-analysis was performed of all the literatures germane to estimate the clinical risk factors of patients with SLE complicated with IFI from published clinical trials from 1990 to April 2022. Mean differences, odds ratio and 95% confidence intervals were calculated, and the meta-analysis was conducted with Stata 12.0 software (StataCorp, College Station, TX). Results: A total of 14 clinical research involving 1129 patients were included. The results of meta-analysis demonstrated that immunosuppressant, glucocorticoids, systemic lupus erythematosus disease activity index score, antibiotic were risk factors associated with IFI in SLE patients. However, age, sex, course of disease, leukopenia, lymphopenia, C- reactive protein and hypoproteinemia were not the risk factors associated with IFI in patients with SLE. Conclusion: Our results indicate that immunosuppressant, glucocorticoids, systemic lupus erythematosus disease activity index score, antibiotic were risk factors for IFI in SLE patients. However, high quality of multicenter, large sample size-controlled trials are needed to validate the result.


Introduction
Systemic lupus erythematosus (SLE) is a common autoimmune disease which characterized by a loss of self-tolerance, the production of autoantibodies and inflammation of multiple organs. SLE has a wide variety of clinical symptoms, which involve multiple organs: skin, serosa, joints, kidneys, central nervous system, and so on. There are a variety of autoantibodies in the patient, which affects not only humoral immunity, but also cellular immunity, and changes in the complement system. The pathogenesis is mainly due to the formation of immune complexes. The exact cause is unknown. The disease showed an alternating process of recurrence and remission. The disease is more common in young women. In Global, the prevalence of SLE in adults range from 30 to 150 per 100,000, and incidence ranges from 2.2 to 23.1 per 100,000 per year. [1] Currently, glucocorticoids (GCs) and/or immunosuppressant are widely used in the treatment of SLE. Those treatment can improve the clinical response rate and survival time. However, due to the widespread use of GCs and immunosuppressants and immune disorders caused by the disease itself, the secondary infection is one of the main complications and main causes of death in patients with SLE, with a mortality rate of about 30%. [2] Invasive fungal infection (IFI) has become an increasingly serious problem, with the prevalence of IFIs varying from 0.83% [3] to 4.8% in different populations. [4] It is usually caused by different types of fungi, such as Cryptococcus, Aspergillus, Candida, histoplasmosis. [5] At present, there are a large number of clinical studies on SLE concurrent fungal infection. Most of researches reported that a systemic lupus erythematosus disease activity index (SLEDAI) 2K score higher than 8 points, the increase of titer of anti-DNA antibody, neutropenia, Medicine lymphocytopenia, use of steroid and antibiotic, mechanical ventilation and hemodialysis have some correlation with IFI. [6] However, a retrospective study by Weng [7] showed that 50% of SLE patients with IFI did not have a higher SLEDAI score. Moreover, most of the above studies come from relatively independent single-center studies, and the results of the risk factors  'M' and 'F' stands for male and female respectively; "R" and "C" corresponds to research group and control group respectively; "NS" stands for not stated; ①age; ②sex; ③course of disease; ④ Immunosuppressant; ⑤Glucocorticoids; ⑥ Antibiotic; ⑦ Leukopenia; ⑧ Lymphopenia; ⑨Creactive protein (CRP); ⑩ Hypoproteinemia; ⑪SLEDAI. NOS = Newcastle-Ottawa scale.
of the studies are not completely consistent, and the results of domestic and foreign studies are also different. Therefore, the purpose of this study is to systematically analyze the related risk factors of SLE concurrent IFI through relevant research reports at home and abroad, so as to early prediction, diagnosis and intervention for IFI in clinic.

Methods
We included case-control studies of IFI risk in SLE patients. The outcomes of our meta-analysis were age, sex, course of disease, immunosuppressant, glucocorticoids, antibiotic, leukopenia, lymphopenia, C-reactive protein (CRP), hypoproteinemia, and SLEDAI Score.

Literature search and selection
Searches were applied to the following electronic databases: MEDLINE, EMBASE, and the Cochrane Library, Chinese Biomedical Database, China National Knowledge Infrastructure, WANFANG and Chinese social sciences citation index databases without language restrictions. The search strategy was based on the combinations of "Lupus erythematosus or lupus nephritis or LE" and "fungal infection or IFI or invasive fungal infections or fungus infection." We also modified the terms according to the different databases. Last query was updated on 15 April 2022. References of retrieved articles were cross-searched to identify any studies missed by the electronic search strategies.

Inclusion criteria
Inclusion and exclusion criteria for original studies were as follows: Firstly, only clinical trials of IFI group comparing with non-IFI for SLE were included regardless of follow up, language or publication status. Secondly, the articles must be proven diagnosis of SLE patients with standard criteria of ACR criteriain in 1997. There were also clear diagnostic criteria for fungal infection. Diagnosis of fungal infections: there are signs and symptoms of fungal infections, which have the cause of infection and meet at least one of the following 3 items: Positive blood fungal culture; puncture bone marrow or closed chamber to take liquid microscopy for fungi or fungal culture Positive, or positive antigen reaction; Sputum, urine, stool and local secretions fungus culture more than 2 times to detect the same fungus. Moreover, when multiple articles were published by the same authors or institutions, the most recent or informative single article was selected. Thirdly, articles lacking original data for meta-analysis, review articles were excluded.

Data extraction
Our initial selection of all candidate articles was relied on careful screening of their abstracts by 2 independent reviewers, using a standardized data collection form, including the following items: the first author, year of publication, sex, mean or median age, course of disease, immunosuppressant, glucocorticoids, antibiotic, leukopenia, lymphopenia, CRP, hypoproteinemia, and SLEDAI Score and assessment of outcomes.
We manually searched the reference lists of the found articles. We also screened references from the relevant literature, including all of the identified studies, but no additional reviews and editorials. Disagreements were resolved by consensus between the 2 readers. In case of persistent disagreement, the final decision was made by our expert.
The quality of the nonrandomized studies was assessed by using the Newcastle-Ottawa Scale with some modifications to match the needs of this study. [8] The quality of the studies was evaluated by examining 3 items: patient selection, comparability of groups, and assessment of outcome. Studies were graded on an ordinal star scoring scale with higher scores representing studies of higher quality. A study can be awarded a maximum of 1 star for each numbered item within the selection and exposure categories and a maximum of 4 stars can be given for the comparability of the 2 groups. The quality of each study was graded as either level 1 (0 to 5) or level 2 (6 to 9).

Statistical methods
All statistical analyses were performed using Statistical Analysis System software (Stata 12.0; StataCorp, College Station, TX), and the P value for the overall effect < .05 with 2-tailed was considered statistically significant. The heterogeneity of all involved studies was assessed by I 2 . When it was lower than 50%, the studies with an acceptable heterogeneity were considered, and then the fixed-effects model with Mantel-Haenszel method was used, otherwise, a random effect model with the Der Simonian and Laird method was adopted. The combined odds ratio (OR) and Mean difference (MD) were initially estimated using Forrest plots. For each trial, the OR and MD was estimated from the original article. If it is not available, we looked at the total numbers of events and the numbers of patients at risk in each group to determine the OR and MD estimate.
Assessment of publication bias was investigated for each of the pooled study groups mainly by the Egger linear regression test. As supplement approach, the Begg rank correlation also was applied to assess the potential publication bias, when P < .05 was considered that there was no publication bias in the study.

Literature search and eligible studies
A total of 253 references were retrieved for initial review using search strategies as described. Seventy-nine citations were excluded from analysis after the first screening based on abstracts or titles. After exclusion of the articles that were out of the scope of our meta-analysis, we identified 17 potential studies for detail evaluation. Upon further review, 2 studies had no control group, and the diagnostic criteria of 1 study was unclear. Finally, 14 studies were performed on the IFI group versus non-IFI in patients with SLE. Selection process for the studies included in the meta-analysis were summarized in Figure 1. The main features of eligible studies in our meta-analysis were summarized in Table 1.

Course of disease.
Six studies [5,[9][10][11]13,18] reported course of disease in patients with IFI compared with controls. There was no statistical heterogeneity among the studies (P = .30, I 2 = 18.3%). Thus, the fixed-effect model was used for statistical analysis. Course of disease was not the risk factors associated with IFI in SLE patients (OR = 0.03, 95% CI −0.14, 0.20, P = .73).

SLEDAI score. Eight studies
And we divided it into 2 subgroups according to the race. SLEDAI score in American was risk factor associated with IFI in patients with SLE (OR = 1.07, 95% CI 0.56, 1.58, P < .001). However, SLEDAI score in Asian was not risk factor associated with IFI in patients with SLE (OR = 0.66, 95% CI −0.09, 1.41, P = .09) (Fig. 7). Summary results for the 2 groups were summarized in Table 2.

Fungal infection strain and site.
Summarizing all the fungal infection strains and parts in all the literature, the most common top 5 strains are Candida albicans, Cryptococcus neoformans, Aspergillus, Candida tropicalis, and others. The top 5 infection sites are oral cavity, lung, gastrointestinal tract, skin, and central nervous system.

Sensitivity analysis and publication bias
The influence analyses revealed that none of the studies significantly affected the pooled MDs, OR or CIs. When each study was sequentially removed and meta-analysis was repeated with the remaining studies, the pooled MD and OR remain nearly the same. Egger linear regression test and Begg test were used to examine publication bias. There was no evidence of publication bias for the analyses of various indicators (e.g., Funnel plot of sex about SLE with IFI compared with those without IFI, Fig. 8).

Discussion
SLE is a kind of diffuse connective tissue disease with multiple organ involvement, which is mediated by autoimmunity and characterized by immune inflammation. Fungal infections can be seen in people whose immune system is unable to remove the invading pathogens or opportunistic infections caused by the imbalance of flora. Among them, deep fungal infections are mostly secondary to diseases with extremely low immune function or even defects. Due to the damage of this disease and the use of immunosuppressive, the patient is in an immunosuppressive state, which is easy to be complicated by IFI. [22] IFI has the characteristics of insidious onset and atypical clinical manifestations. It is easily covered up by the symptoms and signs of the primary disease, especially when it is complicated by bacterial infections. IFI often occurs early in the course of SLE, accounting for between 0.64% and 3.24% of SLE hospitalized patients. [23] These infections resulted in significant mortality, and many were only diagnosed postmortem. In this meta-analysis, 11 possible risk factors were analyzed. Part of the study results are consistent with previous clinical analysis, such as age, sex, course of disease, leukopenia, lymphopenia, CRP, and hypoproteinemia are not risk factors for SLE complicated by IFI; SLEDAI score, the use of GCs, immunosuppressant and antibiotics are risk factors for SLE complicated by IFI.
It is worth noting that in this analysis, we divide the GCs into 3 subgroups and found that the use of GCs ≥ 15 mg and ≥ 45 mg were risk factors. The reason may be that GCs can weaken the body's inflammatory response, affect the redistribution of circulating lymphocytes and lymphocyte function, make the cellular immune function abnormal, reduce the synthesis of immune globulin and complement, and thus affect the body's defense mechanism. However high-dose pulse of GCs is not a risk factor. The specific reason and mechanism are not yet clear, but in clinical work, we must carefully interpret and use high-dose pulse of GCs, and have good predictability and control of disease treatment, outcome, and prognosis.
The analysis of the SLEDAI score shows the difference between races. SLEDAI is a risk factor in American, but it is not in Asian. This may be related to geographical, ethnic, genetic weight, lifestyle, and other factors.
Studies have shown that patients with overlapping IFI and active SLE are more likely to have leukopenia (40%) or normal leukocyte count (42%) than leukocytosis (17%). [23] And this overlap may lead to a higher SLEDAI score. There are also reports that the leukocytes > 3 × 10 8 /L is risk factors affecting secondary fungal infection in SLE. [24] However, the results of this systematic analysis show that leukopenia is a non-risk factor for SLE complicated with IFI. In addition, lymphopenia, C-reactive protein and hypoproteinemia are also non-risk factors in SLE complicated with IFI. This is inconsistent with the results of some studies. The research by Lao [25] show that lymphopenia (OR = 3.28, 95% CI 1.29-8.38, P = .01) was associated with fungal infection in patients with connective tissue disease. This may be related to the location and species of fungi in SLE patients. Therefore, more samples are needed to confirm such inconsistent results.
In addition, we find that immunosuppressant and antibiotics are risk factors for SLE complicated by IFI. Therefore, in the future treatment, it is necessary to pay attention to reducing the use of antibiotics and immunosuppressive agents for SLE patients with potential IFI infection risks.
Due to disease activity, GCs and immunosuppression in SLE patients. The application of fungicides can significantly increase the probability of fungal infection and superficial fungal infection. Oral infection is the most common, while pulmonary infection is the most common. The common pathogen is Candida albicans. SLE combined infections are mainly mixed infections, [26] fungal infections can exist alone, or can be mixed with pathogens such as bacteria and viruses. In the clinical process, according to age, disease course, white blood cell count, neutrophil percentage, 1, 3-β-D-Glucan test (G test), GM (galactomannan) test, full set of virus, purified protein derivative skin test, T-SPOT testing, imaging examination, etiological examination, and other data to identify. In addition to SLE treatment, we need to actively prevent fungal infection. Early diagnosis is needed for suspected cases of infection.
There are some limitations in this study. First, some heterogeneity was observed, but we used subgroup analysis and gave a more reasonable explanation to the results. Second, because of the incorporated literature, including related research at home and abroad, there may be differences in ethnicity, regional culture, and the measurement methods of the same outcome. In addition, some patients with childhood-onset SLE and